Silent Information Regulator 1 Negatively Regulates Atherosclerotic Angiogenesis via Mammalian Target of Rapamycin Complex 1 Signaling Pathway.

This study aimed to investigate the interactions between silent information regulator 1 (SIRT1) and mammalian target of rapamycin (mTOR) in intraplaque angiogenesis and their potential mechanisms through in vivo and in vitro studies. An atherosclerosis model was established in 12 rabbits on a high-cholesterol diet. The rabbits were equally divided into 3 groups: a control group (high-lipid diet), RAP group (high-lipid diet supplemented with rapamycin) and RAP + NAM group (high-lipid diet supplemented with rapamycin and nicotinamide). At the end of 4 weeks, the area of plaques in the aorta was determined and the protein expression of CD31 and vascular endothelial growth factor (VEGF) was detected through hematoxylin and eosin staining and immunohistochemical staining, respectively. For in vitro study, a hypoxia model was established in human umbilical vein endothelial cells (HUVECs) by using the chemical method (CoCl2). The MTT assay, scratch assay and tube formation assay were performed to evaluate the proliferation and angiogenesis abilities of HUVECs. Reverse transcription polymerase chain reaction was used to examine the mRNA levels of SIRT1, hypoxia-inducible factor-1α (HIF-1α), mTOR and p70 ribosomal S6 kinase (p70S6K). Western blotting was used to examine the protein levels of SIRT1, HIF-1α, mTOR, p-mTOR, p-raptor and p-p70S6K. The results of the in vivo study indicated a significant inhibitory effect of rapamycin on plaque size and intraplaque angiogenesis (0.05 ± 0.02mm2 versus 5.44 ± 0.50mm2, P < 0.05). This effect was attenuated by nicotinamide (0.76 ± 0.15mm2 versus 0.05 ± 0.02mm2, P < 0.05). Compared with the RAP group, CD31- and VEGF-positive vessels were abundant in the RAP + NAM group. The RAP group showed lower expression of p-mTOR, p-p70S6K and HIF-1α than did the control group (P < 0.05), whereas the RAP + NAM group showed slightly higher expression of these factors than did the RAP group (P < 0.05). Furthermore, in vitro studies revealed that the inhibitory effect of rapamycin on the angiogenic ability of HUVECs and its significant inhibitory effects on the protein level of HIF-1α and the phosphorylation of proteins involved in the mTORC1 pathway, including mTOR, raptor and p70S6K (P < 0.05), were enhanced by cotreatment with SRT1720 and rapamycin (P < 0.05). In contrast to mTOR and SIRT1, the mRNA levels of p70S6K and HIF-1α were reduced by rapamycin (P < 0.05) and further reduced by cotreatment with SRT1720 and rapamycin. The study results indicate that SIRT1 might negatively regulate atherosclerotic angiogenesis via mTORC1 and HIF-1α signaling pathway and cointervention of SIRT1 and mTOR may serve as a crucial therapeutic strategy in cardiovascular medicine.

Chen R ，Huang Z ，Wang J ，Chen X ，Fu Y ，Wang W ... -  《-》

DEPTOR Deficiency-Mediated mTORc1 Hyperactivation in Vascular Endothelial Cells Promotes Angiogenesis.

The mechanistic target of rapamycin (mTOR) signaling pathway is essential for angiogenesis and embryonic development. DEP domain-containing mTOR-interacting protein (DEPTOR) is an mTOR binding protein that functions to inhibit the mTOR pathway In vitro experiments suggest that DEPTOR is crucial for vascular endothelial cell (EC) activation and angiogenic responses. However, knowledge of the effects of DEPTOR on angiogenesis in vivo is limited. This study aimed to determine the role of DEPTOR in tissue angiogenesis and to elucidate the molecular mechanisms. Cre/loxP conditional gene knockout strategy was used to delete the Deptor gene in mouse vascular ECs. The expression or distribution of cluster of differentiation 31 (CD31), vascular endothelial growth factor (VEGF) and hypoxia inducible factor-1 alpha (HIF-1α) were detected by immunohistochemical staining or western blot. Tube formation assay was used to measure angiogenesis in vitro. Deptor knockdown led to increased expression of CD31, VEGF and HIF-1α in heart, liver, kidney and aorta. After treatment with rapamycin, their expression was significantly down regulated. In vitro, human umbilical vein endothelial cells (HUVECs) were transfected with DEPTOR-specific small interfering RNA (siRNA), which resulted in a significant increase in endothelial tube formation and migration rates. In contrast, DEPTOR overexpression markedly reduced the expression of CD31, VEGF and HIF-1α. Our findings demonstrated that deletion of the Deptor gene in vascular ECs resulted in upregulated expression of CD31 and HIF-1α, and further stimulated the expression of VEGF which promoted angiogenesis, indicating that disruption of normal angiogenic pathways may occur through hyperactivation of the mTORC1/HIF-1α/VEGF signaling pathway.

Ding Y ，Shan L ，Nai W ，Lin X ，Zhou L ，Dong X ，Wu H ，Xiao M ，Zhou X ，Wang L ，Li T ，Fu Y ，Lin Y ，Jia C ，Dai M ，Bai X ... -  《-》

Liquiritigenin inhibits serum-induced HIF-1α and VEGF expression via the AKT/mTOR-p70S6K signalling pathway in HeLa cells.

Liquiritigenin (LQ) is a non-toxic dietary flavonoid with chemopreventive and anticancer properties. However, the mechanism of its antiangiogenesis remains unclear. Hypoxia-inducible factor-1α (HIF-1α) and its downstream target, vascular endothelial growth factor (VEGF), play a critical role in tumour angiogenesis and represent an attractive chemotherapeutic target. In this study, we investigated the effect of LQ on the molecular mechanism of angiogenesis. We found that LQ inhibited VEGF expression at both mRNA and protein levels. Liquiritigenin did not affect HIF-1α expression at the mRNA level, but it dramatically inhibited both serum- and mimicked hypoxic-induced HIF-1α protein accumulation in HeLa cells. Furthermore, we showed that LQ inhibited serum-induced expression of HIF-1α by reducing its stability and decreased the synthesis in a dose-dependent manner. Mechanistically, we demonstrated that LQ inhibited HIF-1α and VEGF expression involved in blocking the protein kinase B (PKB/Akt) signalling pathway, and the mechanisms correlated with dephosphorylation of the mammalian target of rapamycin (mTOR) and its effector ribosomal protein S6 kinase (p70S6K). In addition, LQ inhibited VEGF-induced formation of capillary-like structures in human umbilical vein endothelial cells (HUVEC). Taken together, our study provided valuable insights into the mechanism of antiangiogenic effect of LQ.

Xie SR ，Wang Y ，Liu CW ，Luo K ，Cai YQ ... -  《-》

Imperatorin suppresses proliferation and angiogenesis of human colon cancer cell by targeting HIF-1α via the mTOR/p70S6K/4E-BP1 and MAPK pathways.

Angelica dahurica is a commonly used traditional Chinese medicine to treat migraine headache, toothache and cancer. Imperatorin is an active natural furocoumarin component originating from Angelica dahurica and has been shown to exhibit multiple bioeffector functions, including anti-cancer activity. However, the mechanism by which imperatorin inhibits tumor growth is not fully understood. The aim of this study was to investigate the effectiveness of imperatorin as a treatment of cancer and to identify the underlying mechanisms of its anticancer activity. HCT116, HeLa, and Hep3B cells were used in this study. Major assays were promoter-reporter gene assay, MTT, western blot analysis, immunofluorescence assay, reverse transcription-PCR (RT-PCR), flow cytometric analysis, clonogenic assay, EdU labeling and immunofluorescence, xenografted assay, and VEGF ELISA. We here demonstrated the effect of imperatorin on hypoxia-inducible factor-1 (HIF-1) activation. Imperatorin showed a potent inhibitory activity against HIF-1 activation induced by hypoxia in various human cancer cell lines. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently, whereas it did not affect the expressions of HIF-1β and topoisomerase-I (Topo-I). Further analysis revealed that imperatorin inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), eIF4E binding protein-1 (4E-BP1), eukaryotic initiation factor 4E (eIF4E), extracellular signal-regulated kinase-1/2 (ERK1/2), SAPK/JNK and p38 were significantly suppressed by imperatorin. Furthermore, imperatorin prevented hypoxia-induced expression of HIF-1 target genes and flow cytometric analysis indicated that imperatorin induced G1 phase arrest in human colon cancer cell (HCT116). We found that imperatorin administration inhibits tumor growth and blocks tumor angiogenesis in a xenograft tumor model. These results show that imperatorin inhibited HIF-1α protein synthesis by downregulating the mTOR/p70S6K/4E-BP1 and MAPK pathways. These conclusions suggest that imperatorin is an effective inhibitor of HIF-1 and provide new perspectives into the mechanism of its anticancer activity.

Mi C ，Ma J ，Wang KS ，Zuo HX ，Wang Z ，Li MY ，Piao LX ，Xu GH ，Li X ，Quan ZS ，Jin X ... -  《-》

Tanshinone IIA inhibits HIF-1α and VEGF expression in breast cancer cells via mTOR/p70S6K/RPS6/4E-BP1 signaling pathway.

Li G ，Shan C ，Liu L ，Zhou T ，Zhou J ，Hu X ，Chen Y ，Cui H ，Gao N ... -  《PLoS One》